Ca2+-activated potassium channels, such as small- and intermediate-conductance K+ channels (SK and IK), are widely expressed in excitable tissues. They play pivotal roles in regulating membrane excitability by Ca2+. Unlike voltage-gated K+ channels, activation of SK/IK channels is achieved exclusively by Ca2+. Calmodulin (CaM), tethered to the channel C-terminus, serves as the high-affinity Ca2+ sensor. Four EF-hands, two located at the CaM N-terminus (N-lobe) and the other two at the C-terminus (C-lobe), are the high affinity Ca2+ binding sites. The Ca2+-mediated interaction between CaM and the CaM binding domain (CaMBD) activates the channel. In addition to their physiological roles, SK/IK channels have been implicated in clinical abnormalities. Consequently, a tremendous effort has been devoted to developing small molecules targeting SK/IK channels. While Ca2+-dependent formation of this 2x2 complex is a critical initial step for Ca2+-dependent activation of SK channels, little progress has been made on how binding of Ca2+ to CaM is coupled to eventual opening of the SK channel. SK channels are subjected to regulation by second messengers, most notably; phosphorylation of CaM at T79 by protein kinase CK2 reduces the Ca2+ sensitivity for channel activation. Until now, it remains unknown how phosphorylation of CaM at T79 results in inhibition of SK channels. Phosphoinositides (PIs) play a major role in cellular signaling. PI lipids, particularly PI(4,5)P2, can regulate the channel activities through their direct interactions with the channel proteins, including Kvs, Kir, KCNQ and Cav channels. However, it is virtually not known whether/how PI lipids may regulate SK channel activities We will use integrated approaches of structural biology, computational biology, molecular biology, biophysics and electrophysiology to address these issues, specifically, we will address the following questions: (1) Structural insight into the coupling of Ca binding to CaM and mechanical opening of SK channels. (2) Regulation of the SK channel activity by the membrane lipid, PI(4,5)P2. (3) Regulation, by protein phosphorylation, of the PIP2 affinity for its target proteins. (4) Structural determination of the entire SK channel with or without CaM. Results from the proposed work will provide insights into the molecular mechanisms underlying activation of SK channels by CaM, and regulation of the channel gating by PI lipids. Furthermore, our results will show that convergence of different signaling cascades makes regulation of channel activities by PIP2 possible under physiological conditions, by reducing the affinity of PIP2 for the phosphoryalted channel proteins.